Ceramialean epiphytism in an intertidal Gracilaria chilensis (Rhodophyta) bed in southern Chile

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Journal of Applied Phycology 9: 129–135, 1997. c 1997 Kluwer Academic Publishers. Printed in Belgium.

Ceramialean epiphytism in an intertidal Gracilaria chilensis (Rhodophyta) bed in southern Chile Alejandro H. Buschmann, Claudia A. Retamales & Claudia Figueroa Departamento de Acuicultura, Universidad de Los Lagos, Casilla 933, Osorno, Chile (Phone: (56)-64-205274; fax: (56)-64-239517) Received 15 January 1997; revised 14 April 1997; accepted 16 April 1997

Key words: abundance, ceramiales, Chile, epiphytism, Gracilaria, microscopic stages, recruitment

Abstract The cultivation of the agarophytic red alga Gracilaria has become an activity of major importance in several parts of the world. However, Gracilaria cultivation in Chile still faces problems such as epiphytism. We report ceramialean epiphytism abundance, recruitment patterns and the microscopic stages fixed on the Gracilaria thalli in an intertidal bed (Metri bay) of southern Chile. Using a factorial field experiment, we analyze the effect of intertidal elevation and the use of epiphyte free inoculum on the abundance of ceramialean epiphytic algae. This evidence is used to provide management and control recommendations for ceramialean epiphytes. The results show a summer recruitment and increase in abundance of ceramialean epiphytes. A seasonal and spatial pattern of abundance of microscopic stages of this epiphytic algae was also found, showing a significant increase from the apical (new tissues) to the central parts of the thalli (older tissues). Apical tissues are free of epiphytic propagules. The epiphyte-free tissue zone decreases significantly from winter to summer. Also, a significantly higher density of microscopic stages of ceramialean epiphytes was found on the thalli collected in the farming area during the summer at low tidal levels. The experimental results indicated that the abundance of epiphytes was greater at the lower tidal levels than at the higher distribution limits of Gracilaria and it was not possible to control epiphytism by manual cleaning. Introduction The cultivation of the agarophytic red alga Gracilaria has become of major importance in several parts of the world, such as Asia, South America and southern Africa (Santelices & Doty, 1989). Chile is one of the major world producers with a total production of 60 000 wet t during 1992, of which over 80% was produced by cultivation (Buschmann et al., 1995). The rapid development of Gracilaria cultivation techniques is related to desirable biological characteristics, such as the capacity to anchor the thallus in soft substrata, a high regeneration capacity, high growth rates and good agar yield (Santelices et al., 1984, Pizarro, 1986; Santelices & Ugarte, 1987). The development of this activity produced returns to the country in excess of US$ 40 million in 1995, considering both dry seaweed and agar exportations. Nevertheless, dur-

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ing the past years several problems have arisen which require basic research to determine technological solutions. One of these problems is epiphytism by red, brown and green algae (Pizarro, 1986; Gonz´alez et al., 1993; Buschmann et al., 1995). The epiphytism levels found in commercial Gracilaria beds imply lower algal growth rates, increased loss of stocking biomass and production of raw material with lower value due to the presence of the nuisance algae (Kuschel & Buschmann, 1990; Buschmann & G´omez, 1993; Buschmann et al., 1994). Post-harvest epiphyte control methods seem to be in effective in obtaining an epiphytic-free product, as they are time-consuming and cost-demanding (Buschmann et al., 1995). For this reason, farmers prefer preventive methods to manage epiphyte abundance. Fletcher (1995) pointed out several methods that have being used to control epiphyte abundance, such as physical

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130 removal from the host, reducing the light intensity with netting or changing the light quality, drying of culture systems, changing water circulation, preventive chemical methods using hypoclorite solutions, copper based paints, manipulation of pH and nutrient regimes and biological control methods. Most of these methods are only suitable for tank cultures and are difficult to apply successfully in open culture areas, as is the case of Gracilaria in Chile. In open systems, biological control of epiphytes in suspended (Brawley & Fei, 1987) and bottom (Buschmann et al., 1994) cultures have been tested, but its effectiveness on a full commercial scale still needs to be demonstrated. The knowledge accumulated regarding Gracilaria epiphytism has been mainly descriptive, and there is a need for experimental studies on detailed ecological processes that can sustain recommendations to control epiphytes (Fletcher, 1995). In this study, we evaluate the ceramialean epiphytic loads, prevalence of infections, recruitment and microscopic stage abundance patterns in an intertidal Gracilaria chilensis Bird McLachlan et Oliveira bed in southern Chile. We hypothesized that epiphytic loads fluctuate seasonally and that variations can be explained by recruitment and sporeling abundance of ceramialean algae. By using a factorial field experiment, the effect of intertidal elevation and epiphyte-free host inoculum on the abundance of ceramialean epiphytic algae was assessed. These results were used to propose management recommendations regarding the control of epiphytes.

Materials and methods Study site This study was carried out in an intertidal sandy beach at Metri Bay (41 360 S, 72 420 W), 30 km southeast of Puerto Montt (Figure 1). The tidal amplitude at the site ranged between 5 to 7 m, leaving extensive intertidal areas available for the establishment of commercial Gracilaria farms. The study was undertaken in a ca 1 ha experimental farm planted with G. chilensis between 0 to 2.5 m above the mean low water level (MLW). Intertidal cultures systems have a biomass production potential that ranges between 60 to 75 t (wet) ha 1 y 1 if no nuisance invertebrates or epiphytic algae appear in the culture areas (Buschmann et al., 1995). The principal epiphytes described for this area are brown filamentous species, mainly Giffordia sp., which appear in the spring, different groups of

Figure 1. Experimental farming area in Metri, 30 km south-east of Puerto Montt in southern Chile.

ulvoids (Ulva sp., Enteromorpha sp. and Rhizoclonium sp.) and red species belonging to the Ceramiales (two species of Polysiphonia, Ceramium rubrum (Hudson) C. Ag. and Callithamnion sp.). Methods for assessment of macroscopic abundance and recruitment From June 1997 to June 1993, samples were taken at low (0.7–1.0 m above MLW), intermediate (1.4– 1.5 m) and at the highest (above 2.0 m) Gracilaria distributional limits at Metri Bay. Five samples were taken at each tidal level by removing all the harvestable biomass of Gracilaria in a 0.25 m2 quadrat placed randomly along a 20 m transect running parallel to the shore. The harvested Gracilaria was brought to the laboratory and all the epiphytes were removed manually, classified and the ceramialean species pooled together. Both Gracilaria and the epiphytic ceramialean species were wet weighed on a Sartorius balance ( 0.01 g accuracy) to determine the epiphytic load. The prevalence of ceramialean epiphytes, defined as the frequency at which they appear in the field, was also estimated. This variable is important because it provides an indication of the dispersion of the epiphytes within a farm. For measuring the prevalence, direct field observations were undertaken on the presence of ceramialean epiphytes in thirty 0.25 m2 quadrats. The quadrats were laid randomly in a 100-m

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131 transect parallel to the seashore, at the three tidal levels as described above. For measuring the recruitment of ceramialean juveniles, three ceramic plates and three stones were installed on the sandy beach at the mid-intertidal level (between 1.4 to 1.6 m above MLW) each month, during a year. Two types of substrata (ceramic plates and stones) were used as the recruitment success can depend on their selection (Foster & Sousa, 1985). After two months the plates were brought to the laboratory and the ceramialean cover recorded by placing a transparent plastic sheet with 49 randomly placed dots. The period of two months was long enough to allow the macroscopic development of ceramialean recruits, but too short for other colonizing seaweeds to develop and monopolize the substrata. The plates and stones were installed 3 cm above the bottom to minimize sand deposition and avoid the activity of Tegula atra (Lesson), a common gastropod grazer here (Buschmann et al., 1994). Methods for the assessment of microscopic stages To determine the presence of microscopic stages of ceramialean epiphytes on Gracilaria, thalli were collected at the three tidal levels and brought to the laboratory. In the laboratory, 25 thalli from each tidal height (low, mid and high tidal level) with a minimal length of 12 cm were haphazardly selected and observed under a binocular microscope. Thus, it was possible to count the ceramialean microscopic stages attached on one side of the thallus surface. For each observation, all the sporelings present within a segment of 1 mm Gracilaria thallus were counted to obtain the number of germlings per mm of host thallus. As epiphytic algae were absent from the apical portion of the thalli, the distance from the apical end of the thalli to the first point of appearance of epiphytic propagule attached to the thalli was determined. Due to the high variance in the observations, a high replicate number per treatment (n  15) was preferred over a monthly sampling. For this reason, data were collected on two occasions: in winter (June; low epiphyte abundance season) and during the summer (January; high epiphyte abundance season).

els (0.8 and 1.5 m above MLW). Each experimental quadrat (6) measured 1.5  2.0 m but only the center, with a dimension of 1  1 m, was used to minimize border effects. In each quadrat, two plastic tubes were installed and to each of them three bundles of Gracilaria (100 g each) were tied with a plastic string, as described in earlier studies (Santelices & Ugarte, 1987; Buschmann et al., 1995). Poliethylene tubes were placed in the quadrats and prior to their installation, half of the Gracilaria bundles were manually cleaned in order to eliminate all the macroscopic epiphytes. Bundles with and without epiphytes where distributed randomly inside the experimental quadrats. One month after installation and during a three month period, 6 bundles per treatment, were taken to the laboratory. Each sample was separated in 15 random subsamples and in each of these the presence of ceramialean epiphytes was determined under a stereomicroscope. Based on these data, it was possible to determine the frequency of epiphytes for both treatments. The data were then analyzed by using a twoway ANOVA with time as a repeated measurement, using SYSTAT (Wilkinson et al., 1992) after ensuring the normality and homoscedasticity of variances.

Results Macroscopic abundance and recruitment The results show a seasonal abundance variation of ceramialean algae appearing in September and macroscopically disappearing in April–May (Figure 2A). The epiphytic ceramialean load at the higher intertidal levels can be higher (over twice the Gracilaria biomass), but their occurrence is restricted to short periods in late summer as compared to the lower intertidal levels (Figure 2A). The macroscopic prevalence of ceramialean epiphytes varies greatly during the winter and spring, but increases constantly from values around 20% in winter, reaching 100% during the summer and start to decline the following autumn (Figure 2B). The recruitment of ceramialean epiphytes occurred only on the plates installed in the summer months (January to March) (Figure 3). During the rest of the year no ceramialean algae developed on plates or stones.

Experimental methods Microscopic stages A factorial experiment was implemented to establish the effect of macroscopic epiphytes abundance (with and without macroscopic epiphytes) at two tidal lev-

The abundance of microscopic stages of ceramialean epiphytes on Gracilaria increased significantly

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132 Table 1. Summary of ANOVA showing the effects of the distance from the apical tissues (0–2.5; 2.5–5.0; 5.0–7.5 and >7.5 cm), at three tidal levels of the Gracilaria bed (low, mid, high) and two sampling periods (winter, summer). Treatment

d.f.

F-value

Probability

Distance (D) Tidal level (TL) Sampling Periods (SP) D TL TL SP D SP D TL SP

1 2 1 6 2 3 6

8.66 159.62 775.66 0.76 171.82 1.32 1.36

P